Hye Hyeon Lee

Apoptosis induction of human prostate carcinoma cells by cordycepin through reactive oxygen species‑mediated mitochondrial death pathway

Cordycepin is the main functional component of Cordyceps militaris, which has been widely used in oriental traditional medicine. This compound has been shown to possess many pharmacological properties, such as enhancing the bodys immune function, and anti-inflammatory, anti-aging and anticancer effects. In the present study, we investigated the apoptotic effects of cordycepin in human prostate carcinoma cells. We found that treatment with cordycepin significantly inhibited cell growth by inducing apoptosis in PC-3 cells. Apoptosis induction of PC-3 cells by cordycepin showed correlation with proteolytic activation of caspase-3 and -9, but not caspase-8, and concomitant degradation of poly (ADP-ribose) polymerases, collapse of the mitochondrial membrane potential (MMP). In addition, cordycepin treatment resulted in an increase of the Bax/Bcl-2 (or Bcl-xL) ratio, downregulation of inhibitor of apoptosis protein (IAP) family members, Bax conformational changes, and release of cytochrome c from the mitochondria to the cytosol. The cordycepin-induced apoptosis was also associated with the generation of intracellular reactive oxygen species (ROS). However, the quenching of ROS generation with antioxidant N-acetyl-L-cysteine conferred significant protection against cordycepin-elicited ROS generation, disruption of the MMP, modulation of Bcl-2 and IAP family proteins, caspase-3 and -9 activation and apoptosis. This indicates that the cellular ROS generation plays a pivotal role in the initiation of cordycepin-triggered apoptotic death. Collectively, our findings suggest that cordycepin is a potent inducer of apoptosis of prostate cancer cells via a mitochondrial-mediated intrinsic pathway and that this agent may be of value in the development of a potential therapeutic candidate for both the prevention and treatment of cancer.

Anti-inflammatory effects of cordycepin in lipopolysaccharide-stimulated RAW 264.7 macrophages through Toll-like receptor 4-mediated suppression of mitogen-activated protein kinases and NF-κB signaling pathways

Cordycepin is the main functional component of the Cordyceps species, which has been widely used in traditional Oriental medicine. This compound possesses many pharmacological properties, such as an ability to enhance immune function, as well as antioxidant, antiaging, and anticancer effects. In the present study, we investigated the anti-inflammatory effects of cordycepin using a murine macrophage RAW 264.7 cell model. Our data demonstrated that cordycepin suppressed production of proinflammatory mediators such as nitric oxide (NO) and prostaglandin E2 by inhibiting inducible NO synthase and cyclooxygenase-2 gene expression. Cordycepin also inhibited the release of proinflammatory cytokines, including tumor necrosis factor-alpha and interleukin-1-beta, through downregulation of respective mRNA expression. In addition, pretreatment with cordycepin significantly inhibited lipopolysaccharide (LPS)-induced phosphorylation of mitogen-activating protein kinases and attenuated nuclear translocation of NF-κB by LPS, which was associated with abrogation of inhibitor kappa B-alpha degradation. Furthermore, cordycepin potently inhibited the binding of LPS to macrophages and LPS-induced Toll-like receptor 4 and myeloid differentiation factor 88 expression. Taken together, the results suggest that the inhibitory effects of cordycepin on LPS-stimulated inflammatory responses in RAW 264.7 macrophages are associated with suppression of mitogen-activating protein kinases and activation of NF-κB by inhibition of the Toll-like receptor 4 signaling pathway.

Cordycepin increases sensitivity of Hep3B human hepatocellular carcinoma cells to TRAIL-mediated apoptosis by inactivating the JNK signaling pathway

Resistance to tumor necrosis factor-related apoptosis‑inducing ligand (TRAIL)-induced apoptosis has been reported in various cancer cells. Cordycepin, a specific polyadenylation inhibitor, is the main functional component in Cordyceps militaris, which possesses many pharmacological activities including antitumor and anti-inflammation. In the present study, we demonstrated that treatment of cordycepin sensitized TRAIL-resistant Hep3B human hepatocellular carcinoma cells to TRAIL-mediated apoptosis as evidenced by formation of apoptotic bodies, chromatin condensation and accumulation of cells in the sub-G1 phase. The induction of apoptosis following co-treatment with cordycepin and TRAIL in Hep3B cells appeared to be correlated with modulation of Bcl-2 family protein expression and activation of the caspase cascade, which resulted in the cleavage of poly(ADP-ribose) polymerase and β-catenin. In addition, cordycepin treatment also inhibited activation of c-Jun N-terminal kinase (JNK). Pretreatment with SP600125, a JNK inhibitor, resulted in a significantly increased sub-G1 population and caspase activity in cordycepin plus TRAIL-mediated apoptosis. Taken together, these results indicate that JNK acts as a key regulator of apoptosis in response to combined treatment with cordycepin and TRAIL in human hepatocellular carcinoma Hep3B cells.

Mori folium inhibits interleukin-1β-induced expression of matrix metalloproteinases and inflammatory mediators by suppressing the activation of NF-κB and p38 MAPK in SW1353 human chondrocytes

The pro-inflammatory cytokine interleukin-1β (IL-1β) is known to play a crucial role in the pathogenesis of osteoarthritis (OA) by stimulating several mediators that contribute to cartilage degradation. Mori folium, the leaves of Morus alba L., has long been used in traditional medicine to treat diabetes, protect the liver, and lower blood pressure; however, the role of Mori folium in OA is not yet fully understood. Therefore, in the present study, we investigated whether Mori folium water extract (MF) inhibited the catabolic effects of IL-1β in vitro, and also whether it inhibited the matrix metalloproteinases (MMPs), inducible nitric oxide (NO) synthase (iNOS) and cyclooxygenase-2 (COX-2) through the attenuation of nuclear factor-κB (NF-κB) and mitogen activated protein kinase (MAPK) pathways in SW1353 human chondrocytes. MMP proteins in culture medium were determined using a cytokine‑specific enzyme-linked immunosorbent assay (ELISA). The production of NO and prostaglandin E2 (PGE2) were evaluated using Griess reagent and ELISA. Subsequently, the mRNA and protein levels of MMPs, iNOS, COX-2, NF-κB and MAPKs were examined by RT-qPCR and/or western blot analysis. The results indicate that MF significantly reduced the IL-1β‑induced release of MMP-1 and -13 in SW1353 cells, which was associated with the inhibition of MMP-1 and -13 mRNA and protein expression in a concentration‑dependent manner at concentrations with no cytotoxicity. MF also attenuated the IL-1β-induced production of NO and PGE2, and reduced iNOS and COX-2 expression. Furthermore, we noted that MF markedly suppressed the IL-1β‑induced nuclear translocation of NF-κB, which correlated with the inhibitory effects of MF on inhibitor-κB (IκB) degradation, and the phosphorylation of p38 MAPK was selectively restored by MF upon IL-1β stimulation. These results indicate that MF inhibited the production and expression of MMP-1 and -13 and inflammatory mediators, at least in part, through suppressing the activation of either NF-κB or p38 MAPK in IL-1β-treated SW1353 chondrocytes. Therefore, the novel findings of the present study suggest that MF is a potential therapeutic choice for chondroprotection against the collagen matrix breakdown in the cartilage of diseased tissues, such as those found in patients with arthritic disorders.

Schisandrae Fructus Inhibits IL-1β-Induced Matrix Metalloproteinases and Inflammatory Mediators Production in SW1353 Human Chondrocytes by Suppressing NF-κB and MAPK Activation

Preclinical Research

Purification and biochemical characterization of a 17 kDa fibrinolytic enzyme from Schizophyllum commune

A fibrinolytic enzyme of the mushroom, Schizophyllum commune was purified with chromatographic methods, including a DEAE-Sephadex A-50 ion-exchange column and gel filtrations with Sephadex G-75 and Sephadex G-50 columns. The analysis of fibrin-zymography and SDS-PAGE showed that the enzyme was a monomeric subunit that was estimated to be approximately 17 kDa in size. The fibrinolytic activity of the enzyme in plasminogen-rich and plasminogen-free fibrin plates was 1.25 and 0.44 U/ml, respectively. The N-terminal amino acid sequence of the purified enzyme was identified as HYNIXNSWSSFID, which was highly distinguished from known fibrinolytic enzymes. The relative activity of the purified enzyme with an addition of 5 mM EDTA, Phosphoramidon, and Bestatin was about 76, 64, and 52%, respectively, indicating that it is a metalloprotease. The optimum temperature for the purified enzyme was approximately 45°C, and over 87% of the enzymatic activity was maintained as a stable state in a pH range from 4.0 to 6.0. Therefore, our results suggest that the potential thrombolytic agent from S. commune is a unique type of fibrinolytic enzyme.

Effect of Fermented Cudrania tricuspidata Fruit Extracts on the Generation of the Cytokines in Mouse Spleen Cells

We investigated a physiological function by fermenting a medicinal mushroom, (Cudrania tricuspidata fruit). A fermentation using lactic acid bacteria and the extracts isolated from 70% ethanol fractionation was included in cultured mouse spleen cells for cytokine secretion. As a result, total polyphenol content improved by 47% by organic acid fermentation. This was regarded as immune activity in fermented C. tricuspidata fruits, as the levels of interleukin (IL)-2 and IL-4 secretion increased. In addition, when the extracts were treated with a stimulant lipopolysaccharide, the secretion of helper T (Th) 1 cytokines IL-2, IL-12, and tumor necrosis factor-α was suppressed, while the secretion of Th2 cytokines IL-4, IL-5, IL-6, and IL-10 significantly increased. Therefore, this study suggests that fermentative C. tricuspidata fruit extracts can contribute to the suppression of cellular immune reactions induced by the expression of Th1 cells and activation of the expression of Th2 cells inducing humoral immune reactions associated with the antibody generation by B lymphocytes.

Effect of Black Garlic Extract on Cytokine Generation of Mouse Spleen Cells

생리활성물질을 다량함유하고 있는 마늘의 발효산물인 흑마늘의 면역활성을 검증하기 위하여 C57BL6 마우스 비장세포를 이용하여 흑마늘이 비장세포의 활성화에 미치는 영향을 확인하였다. 흑마늘 추출물은 시판되는 남해 흑마늘 액기스를 농축하여 사용하였다. 그 결과 IL-2에서 흑마늘 추출물만 처리한 군에서 생성이 증가하였으며, LPS와 흑마늘 추출물을 함께 처리하였을 때 IL-2와 TNF-

Biochemical analysis of a fibrinolytic enzyme purified from Bacillus subtilis strain A1

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Initial acidic pH is critical for mycelial cultures and functional exopolysaccharide production of an edible mushroom, Laetiporus sulphureus var. miniatus JM 27

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